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This paper was submitted by the faculty of FAU’s Harbor Branch Oceanographic Institute.

Notice: ©2000 Springer‐Verlag. This manuscript is an author version with the final publication available at http://www.springerlink.com and may be cited as: Pomponi, S. A., & Willoughby, R. (2000). Development of cell cultures for biomedical application. In C. Mothersill & B. Austin (Eds.). Aquatic invertebrate cell culture. (pp. 323‐336). Berlin: Springer‐Verlag.

14

Development of sponge cell cultures for biomedical application

Shirley A. Pomponi and Robin Willoughby

14.1 IMPORTANCE OF SPO GE CELL CULTURE

The mari ne environment is a rich source of both biological and chemical diversity. During the past two deca des, there has been significant effort made in the discovery of novel, marine-derived, nat ural products with po tential for development as phar­ maceut icals, nut ritional supplements, cosmetics, agr ichemicals, molecular probes, enzymes and fine chemicals. Each of these classes of marine bioproducts has a potential multibillion-dollar mark et value (BioScience, 1996). (phylum Porifera) have been the most studied group for mari ne biopro­ ducts (Munro et al., 1999) and have yielded the greatest number of compo unds (I reiand et al., 1993). The emphasis on discovery of biolo gically active sponge metabolites is due to a number of factors. Sponges are among the most abunda nt and diverse groups of invertebrates present in benthic marine environment s world­ wide. There are ", 6,000 described species of sponges and perhaps twice as many undescribed species. Although sponge l arvae ~ 'pr~nktonic , adult sponges are sessile. It is not so diffieult to understand why sponges are a major source of cytoto xic, antibiotic, and anti-inflammatory compounds. During the course of their evolutionary history, they have evolved the abilit y to biosynthesize metabolites for defense against predati on , for inhibition of settlement or overgrowth by compet­ ing organisms, for contro l against infection by the microbial flora that filter through their bodies, for reproductive cues to conspecifics and for recognition of self and non- self (i.e. immune responses). Unfortunately, the roles that most biologically active metabolites play in the marine organisms that synthesize them are largely unknown. An und erstanding of the roles of these metabolites in nature could, in fact, lead to a more rational approach to the discovery of commercially useful marine bioproducts. 324 Development of sponge cell cultures for biomedical applications [Ch.14

As a result of the potential commercial importance of sponge-derived natural products, there has been a renewed effort to understand their phylogenetic relation­ ships, chemical ecology, and physiology in an attempt to predict the occurrence or determine trends in the production of novel secondary metabolites, as well as to understand the mechanisms which stimulate the production of these compounds. In addition, with the awareness that natural populations of sponges (and other marine organisms) cannot support the harvests predicted for supply of sufficient quantities of bioactive compounds for development of the bioproducts, research is in progress to develop alternative supply methods, such as chemical synthesis, aquaculture, cell culture and recombinant production. The objective of research in progress in our laboratory is to establish cell lines of bioactive marine invertebrates that can be used as models to study in vitro produc­ tion of bioactive metabolites and the factors which control expression ofproduction. We hypothesize that understanding the molecular mechanisms involved in growth regulation and bioactive metabolite production in these organisms will lead to the development of genetically engineered cell-lines capable of overexpression of bioac­ tive natural products. Perhaps more important, however, is the development of cell lines of these 'simple' metazoans to study basic cell physiology and molecular biology that may be applied to understanding more complex metazoan systems, including humans.'

14.2 SPONGE CELL CULTURES

14.2.1 Cell culture versus tissue culture Traditionally, cell cultures are differentiated from tissue or organ cultures on the basis of a few key criteria: their lineage, their potential for cell division, and their uniformity (Freshney, 1987). Tissue or organ cultures are derived from explants or fragments of the tissue or organ; they retain the architecture characteristic of that tissue or organ, and they are comprised-of differentiated cells with limited capacity for cell proliferation. Cell cultures may be derived from explants or dissociated cell suspensions; they have the capacity for proliferation, and cells with similar rates of growth will predominate to form lines of homogeneous cell types. Typically, normal (mammalian) cells will form a monolayer and must remain attached to the substrate to proliferate, while only haemopoietic, malignant or transformed cells will grow in suspension (Freshney, 1987). With an increase in the understanding of basic metabolic processes at the cellular level in mammalian cell cultures has come a transition to focusing on understanding these processes in differentiated, three­ dimensional populations of cells. A similar trend is occurring in sponge cell culture. While there is still much to learn about basic cellular and molecular processes in sponge cells, there is much to gain from research using both undifferentiated and differentiated cells in both cell cultures and 'tissue cultures' (e.g. primmorphs, Custodio et al., 1998; Muller et al., 1999; Muller and Custodio, this volume). We have the opportunity to compare differential expression in differentiated versus Sec. 14.3] Establishment of ceU cultures 325 undifferentiated cells, in homogeneous versus heterogeneous/integrated populations and in cell-culture monolayers versus three-dimensional cultures which preserve the architecture of the 'functional' adult sponge . . Because of their cellular level of organization, sponges can be easily dissociated into single cells (Wilson, 1907), which will reaggregate to form a functional sponge (i.e. an aggregate with differentiated cells). This basic ability makes sponges the ideal organism to use as an in vitro model. A cell culture is most likely an equilibrium of multipotent stem cells, undifferentiated but committed precursor cells, and mature differentiated cells. The equilibrium will shift according to changes in culture con­ ditions (Freshney, 1987). The challenge is to understand both the position of the cell in its lineage (i.e. whether it is an uncommitted stem cell, a committed but undiffer­ entiated precursor or a mature differentiated cell) and to understand the conditions that will induce the cells to differentiate or dedifferentiate. For sponge cells, many of these questions remain to be addressed. The development of a cell line may simply appear to involve the application of existing concepts and technology developed for the culture of other heterotrophic eukaryotic cells, such as vertebrate cells (Ham and McKeehan, 1979). The discovery of growth factors, cytokines, and hormones, together with the development of basal nutrient media for specific cell types (Hayashi and Sato , 1976) has made it virtually routine to culture a wide variety of vertebrate cell types. But even in the case of vertebrate cells, culture of specialized cell types beyond primary culture (e.g. epi­ thelial cells) was only achieved by empirical means during the last 30 years (Kaighn and Lechner, 1984). In contrast, very little is known about culture requirements for marine sponges or, for that matter, for marine invertebrates in general. Development of marine invertebrate cell lines (i.e. continuously replicating normal or transformed cells) has been problematic. The status of the science and the problems encountered are reviewed by Rinkevich (1999).

14.3 ESTABLISHMENT OF CELL CULTURES

14.3.1 Selection of appropriate species for in vitro research

.-;- . ~ ~ " .. ~ - I- - If the objective of sponge cell culture is the mvitro production of bioactive metabo­ lites or the evaluation 'of cellular and molecular events involved in the production of these compounds, selection of the appropriate sponge species is essential. A number of secondary metabolites reported from marine invertebrates have been proposed to have a microbial origin. This is generally based upon: (1) the presence of the compounds as trace metabolites in the source organism; and /or (2) close structural similarity to compounds reported either from microbial sources or from widely divergent taxa. However, in only a few cases have the compounds been unequivo­ cally localized in microbial associates (e.g. Unson et al., 1994; Bewley et al., 1996). There are equally compelling data to indicate that bioactive metabolites are localized in sponge cells (Faulkner et al., 1993; Garson et al., 1994, 1998; Uriz et al., 1996a, b; Flowers et al., 1998). 326 Development of sponge cell cultures for biomedical applications leh.14

The prim ary species selected for our cell-culture development research is Teichax­ inella morchella (= corrugata) (Demospongiae: Axinellida). The antitumour compound stevensine (Albitzi and Faulkner, 1985) constitutes approximately 0.5 per cent of the wet weight of this sponge (Pomponi et al.. 1997a). Similar compounds have been found in related axinellid taxa but do not occur randomly in unrelated taxa. A non-microbial origin is further supported by our microscopic examinations, which indicate that there are no associated intra- or intercellular micro-organisms in suffi cient numbers to be responsible for the observed level of production of the metabolite.

14.3.2 Identification of target cell types Sponges contain a variety of cell types. Archaeocytes are totipotent cells that are necessary for attac hment, aggregation and differenti ation (DeSutter and Buscema, 1977), and are capable of differenti ation into other cell types (for a review see Simpson , 1984). Since man y sponge cell types are . terminally differenti ated, we hypoth esized that enriching and selecting for archaeocytes would result in cell cul­ tures capable of proliferati on and expression of bioactive metab olite production. Moreover, experiments to evalua te localization of the antibiotic, 5-hydroxy trypto­ phan (Sennett et al., 1990), produced by Hym eniacidon heliophila (Demos po ngiae: Halichond rida) and the antitum our compound, stevensine (Pomponi et al., 1997b), produced by Teichaxinella morchella, have shown that both compounds are localized in archaeocyte subpopulations.

14.3.3 Selection of appropriate methods for cell dissociation Sponges can be dissociated into cell suspensions using a variety of physical, chemical and enzymatic techniques. The choice will depend on how the dissociated cells will be used. For example, if it is not necessary to obtain a monodisperse suspension or enriched cell subpopulation, mechanical dissociation (e.g. squeezing or mincing the sponge to release cells) is a ccep~Q! e. However, if monodisperse suspensions are necessary, the use of calcium-Tand magnesium-free artificial sea water (CM FSW), chelators'(e.g, EDTA) and/or enzymes (e.g., trypsin) ma y be indicated to prevent the cells from reaggregating. It has been our experience that the use of EDTA and/or trypsin reduces the viability of the cells, as does soaking the cells in CM FSW for longer than 20 min. It is not surprising that cells kept in CMFSW for up to 24 h will not und ergo DNA synthesis (Custodio et al., 1998; Mull er and Custod io, this vol­ ume). Therefore, careful manipulation of the dissociation conditions to achieve disaggregati on, while not irreversibly inhibiting DNA synthesis, is essential. In our laboratory, inocula for cell cultures are prepared by dissocia ting sponge frag ments (cleaned of debri s and rinsed in filtered sea water) into cell suspensions by soaking in CMFSW at a volume of 10: I (CMFSW: spo nge volume) for ID-20 min, and then gently forcing the suspension through sterile gauze to further disperse the cells (Pom po ni and Willoughb y, 1994; Pomponi et al., 1997b). After the cell suspen­ sion is filtered throu gh 70-11m mesh nylon to remove cell aggregates and debris, the Sec. 14.4] Development of defined culture media 327

filtrate is concentra ted to 106-108cellsml- 1 by cent rifugation at 300 x g for 5min. Archaeocy tes are separated from other cell types by layering 2-4 ml of the crude cell suspension on a discrete gradient of osmo tically adju sted Percollj CMFSW and centrifuging at 400 x g for IOmin (Pomponi and Willoughb y, 1994). Th e band s at the Percoll interfaces are collected by aspira tion with a pipette, rinsed by diluting with CMFSW and concentrated by centrifuging (5 min at 300 x g). Viabilit y is assessed by trypan-blue exclusion. In general, bacteria, microftagellates and sponge choa nocytes are retained on the least dense layer. Archaeocytes and spherulous cells are retained at higher density layers. The best gradient for homogeneous separation of the desired cell type must be determined empirically for each species; however, a good starting point is 15-30-45- 60-75 per cent PercolljCM FSW . Ficoll has been used by others (Garson et al., 1994; Uriz et al., 1996a, b;Flowers et al., 1998); however, it is our experience that viability is significantly decreased when cells are enriched using Ficoll gradients.

14.3.4 Cryopreservation: pros and cons Although it is generally preferable to initiate cultures with cells dissociated from freshly collected sponges, this is often not practical because of limited opportunities for field collections. We have developed a meth od for cryopreservation of sponge cells which results in viability comp arable with that obtained with cryopreserved 8c 1 mamm alian cell lines (Pomponi et al., 1997b). Archaeocytes (l07-10 ellsml- ) are suspended in a solution of 20 per cent FCS and 2.3 mM rifampi cin in culture medium . An equal volume of CMFSW containing 15 per cent DMSO is added slowly to the cell suspension, and the mixture is dispensed into 1- 2-ml plastic cryovials. All solutions are sterile and the entire procedure is done on ice. Vials l are cooled at a slow, controlled rate (l °Cmin - ) using Nalgene cryocont ainers at - 70°C for 4 h. Samples are stored at - 140°C or in liquid-nitrogen vapour. When cells are needed for establishment ofcultures, they must be thawed rapidl y in a 50°C water bath, then rinsed in CMFSW. This method was tested on eleven species of sponges. Prior to cryopreservation, archaeocyte viability after dissociation and se­ lective cell enrichment ranged from 68 to 100 per-eenr'depending on the species. After cryopreservation, viabilit y ranged from 62 to 95 per cent (Ta ble 14.1). The availability of this simple freezing procedure permits preservation of a variety of species of both shallow-water and deep-water sponges at the time of collection and enables the preparation of uniform lots of cells to be used for subsequent experi­ ments.

14.4 DEVELOPMENT OF DEFINED CULTURE MEDIA

Since the objective of these studies is not the formation of differentiated, three­ dimensional cultures, but rather the establishment of homogeneou s cell lines, 328 Development of sponge cell cultures for biomedical applications [eh.14

Table 14.1. Viability of arch aeocytes before and after cryopreservation",

Species Before (%) After (%)

Order Halichondrida Hymeniacidon heliophila 100 91 Hal ichondria melanadocia 99 82 Order Axinellida gracilis 68 69 Teichaxinella morchella 95 95 Order Haplosclerida Xestospongia mula 100 88 Order Hadromerida Anthosigmella varians 86 62 Suberites spp. 99 82 Order Poecilosclerida Tedania ignis 98 93 Mycale spp. 85 75 Order Spirophorida Cinachyra spp. 96 82 Order Verongida Aplysina spp. 80 66 a Percent as determined by trypan-blue exclusion.

media-optimization experiments are designed based on the hypothesis that disso­ ciated cells would be able to absorb dissolved organic nutrients. Our objective, therefore, is to develop a defined soluble nutrient medium . We avoided the use of particulate organics (e.g. heat-killed bacteria or phytoplankton) that have been used by other researchers. There are two basic experimental- approaches to developing an optimal nutrient medium f9r cell cultures. In the first, the concentrations of individual constituents of a defined medium are altered until an optimum medium is obtained. The second strategy, which is the approach we used, starts with an existing medium and supple­ ments it with growth factors and trace elements . First, a suitable basal medium that would maintain sponge cell viability was selected. Commercially available media, adjusted to the proper osmolarity with NaCl, were compared by monitoring meta­ bolic activity. We have developed a series of multiwell-plate assays to monitor DNA content (via binding to Hoechst 33342), protein synthesis (using sulphorhodamine B) and esterase activity (using fluorescein diacetate) in primary sponge cell cultures (Pomponi et aI., 1997a; Willoughby and Pomponi, 2000). These assays, in addition to direct microscopic evaluation and counting, as well as flow-cytometric cell-cycle analyses (Pomponi and Willoughby, 1994) were used to monitor effects ofmedia and supplements. Sec. 14.4] Development of defined culture media 329

14.4.1 Basal media Medium 199 (M- 199) (Gibco BRL 11044-013) was selected as the basal medium because it consistentl y supported cell viability in the species tested (Pomponi et al., 1997a). Iscove's MDM (Gibco BRL 21056-015) is a suitable alterna tive basal medium . Osmolality was increased to 1,000 mosmol by addition of NaCl. The pH was adjusted to 8.1 and stabilized by addition of 5 mM Trizma buffer. FCS was added at 5 per cent, but was eliminated from experiments in which the effects of vertebrate growth factors were evaluated. Addition of antibiotics was adjusted accordi l)g to experimental requirements (Pomponi and Willoughby, 1994).

14.4.2 Antibiotics Sponge cell culture presents a unique purification challenge because, unlike higher metazoans , there are no areas of a sponge from which an aseptic inoculum can be obtained. To further complicate establishment of axenic cell cultures, many sponges host endosymbiotic micro-organisms that may be released into an otherwise sterile culture if the cells lyse. While establishment of an axenic sponge cell line is our long­ term objective, our efforts to date have been to control microbial contamination sufficiently to conduct experiments on nutrient and growth requi rement s without interference ofcontaminants. A combination of filtration and use of sterile solutions during cell dissociation, selective sponge cell enrichment by density-gradient separa­ tion and antibiotics to prevent growth of remaining micro-organisms has pro ven to be most effective (Pomponi and Willoughby, 1994; Pomponi et al., 1997a). Rifampi­ cin (1.l6mM) is the most effective at controlling microbi al contamination in all species tested with minimal effect on sponge cell viability (Pomponi and Willoughby, 1994). Keto conazole (9.4 11M) or amphotericin B (Fungizone) (2.7 11M) may be used to inhibit fungal growth. .

14.4.3 Growth factors To stimulate cell division, prim ary cultures of Hymeniacidon heliophila were treated with lectins that are mitogenic in mammalian cultures. Concanavalin A (Con A, 10ug ml"), phytohaemagglutinin (PHA) (1.5 per cent by-VOl i:iilie), lipopolysacchar­ ide (LPS, 10Ilgml -I ) and pokeweed mitogen (PKM, I per cent by volume) were 6 1 tested individually by incubating primary cultures (10 cells ml- ) in sponge cell­ culture medium with lectins added at concentrations reported to be mitogenic in vertebrate cells. After 72 h, twice as many sponge cells, as measured by direct haemocytometer counts, were present in PHA-treated cultures as compared with controls. In addition, synthesis of DNA was confirmed by flow-cytometric cell­ cycle analysis (Pomponi and Willoughby, 1994). Teichaxinella morchella cells stimu­ lated with PHA divided within 36 h, as measured both by direct sponge cell counts (Pomponi et al., 1997b) and by measurement of DNA content (Fig. 14.1). PHA consistently stimulates cell division of every sponge species tested within 36-72 h after inoculation. 330 Development of sponge cell cultures for biomedical applications [eh.14

200

100 -.------.....-...- -.----.--... -- -.---.--.--..--.--.----..--.-.------.....-....---.-.-..-_._--._.-...._ -.._ _..--

O...... --....-----~-----~----_r--- 6 18 30 42 Hours postinoculation

Fig. 14.1. PHA-induced DNA synthesis in Teichaxine/la morche/la cells. DNA was measured by the Hoechst 33342 microtitre plate assay. At 18 h post-inoculation , the DNA content of PHA-stimulated cultures increased. The relative DNA content of PHA- stimulated cultures reached a plateau at 30 h, at greater than 200 per cent of the DNA content of the untreated control (mean ± S.E., n = 5; redrawn from Pomp oni et al., 1997b).

Genes, which code for growth-regulating compounds, have been identified in sponges. Subunits of lectins isolated from , and mitogenic in, Geodia cydonium primary cell cultures have been cloned from a cDNA library of G. cydonium (Pfeifer et al., 1993). Robitzki et al. (1990) demonstrated that the preproinsulin gene is present in G. cydonium, that insulin is secreted by specialized spherulous cells and that it causes gene activation. Our laboratory has demonstrated that insulin induces an increa se in protein concentration in cell cultures of Teichaxinella morchella (Fig . 14.2) (Pomponi et al., 1997b), providing further evidence that sponges have insulin receptors. We have also demonstrated an increase in DNA content of sponge cell cultures as a result of other exogenous growth-regulating compounds, such as EGF, bovine pituitary extract, .~J:IA, prostaglandin E2 (Fig . 14.2) (Pomponi and Willoughby, 1994; Pomporu-:et ol., 1997b), FGF, interleukin-6 and FCS (data not shown). Enhanced DNA content in response to stimulation with FCS is not surpris­ ing, since traces of many growth-regulating compounds may be found in serum. Our eventual goal , however, is to optimize cultures in a totally defined medium.

14.5 SELECTION OF CULTURE VESSEL

14.5.1 Attached versus suspension cultures Dissociated sponge cells do not firmly attach to culture vessels. In fact, it is this characteristic which enables the formation of aggregates and primmorphs when the cultures are gentl y shaken or rot ated (Muller et al., 1999). Although many of our Sec. 14.5) Selection of culture vessel 331

250

200

150

/ 1 00 ------

50

gfc egf bpe aa pha ins pe2

Growth factor Fig. 14.2. Effect of growth factors on Teichaxinella morchella primary cultures after 48 h incubation in Iscove's MOM . Data are plotted as percent control of protein concentra tion, as measured by the SRB assay. Control cultures (horizontal dashed line) received no growth factors. gfc, frowth factor cocktail; egf, epidermal growth factor, lu ng ml" : bpe, bovine pituitary extract, 25 J.1g ml- ; aa, arachidonic acid, 10J.lM ; linoleic acid, 0.2 J.lM ; cholesterol, 5 J.lM ; phosphoethanolaminc, 10 ug ml " , pha, phytohaemagglu­ l l tinin, 1.5 per cent; transferrin, 5 ~lg m l -l ; ins, insulin, 10J.lgml- ; pe2, prostaglandin E2, 50 ng ml- ; hydrocortisone, 50 nM; retinol acetate, 0.3 J.lM . Concentrations of components of growth-factor cocktail tested individually are the same as in the complete gfc (mean ± S.E., n = 5; redrawn from Pomponi et al., 1997b). experiments to evaluate matrices of medium supplement s are necessarily conducted using adh erent cells in multiwell plates or flasks, it is also possible to establish prim ary cultures of cells in suspension. Indeed, eventual scale-up of sponge cell cultures for in vitro production of bioactive metabolites may necessitate the use of suspension cultures. Cryopreserved cells from bioactive species representing broad ranges in taxonomy and depth were used for scale-up experiments in 5Q:ml- shaker flasks: from shallow water the axinellid Ptilocaulis gracilis and Teichaxin ella morchella, and the calcareous sponge Leucetta spp.; from deep water demosponges, Spongosorites spp. (order Halichondrida), Dercitus spp . and Asteropus spp . (order Choristida) and Cribrochalina spp. (order Haplosclerida). Cells in shaker flasks remained viable and did not form large aggregates , but populations were stationary, neither increas ing nor decreasing in number. Cells of Dercitus spp. and Ptilocaulis gracilis were inocu­ lated into IOO-ml spinner flasks with sponge cell-culture medium supplemented with PHA. Medium was changed every 3-4 days and cells were micro scopicaIly counted at each medium change . The Dercitus culture was difficult to monitor for ceIl in­ crease because the cells attached to the impeIler blade of the spinner flask. No attempt was made to dislodge the cells, which would have resulted in cell damage and a decrease in cell number. The patches of attached ceIls (characteristicaIly 332 Development of sponge cell cultures for biomedical applications [Ch.14 pigmented a dark purple, as in the source sponge) increased in area, indicating an overall increase in cell number. After I month, some cells became suspended and periodic subsampling of just the suspended cells indicated one population doubling in 3 days. Spinner-flask cultures of Ptilocaulis gracilis which were stimulated with growth factors and PHA underwent three population doublings in 8 days and were subsequently passaged into 250-ml and 500-ml spinner flasks with no decrease in viability; however, the cells entered a stationary phase after the third doubling. Although suspension culture is, in general, characteristic of only haemopoietic, transformed or malignant mammalian cell lines, it has been suggested that normal stem cells or uncommitted precursor cells also retain this ability (Freshney, 1987). This is an intriguing concept that may be used to select for stem cells from suspen­ sion cultures of sponge cells in the future.

14.6 CELL-TYPE VERIFICATION AND ASSESSMENT OF FUNCTIONAL STATE

Fundamental to the establishment and culture of any cell line is the ability to verify its identity at all stages ofculture. Microbial contamination can be a serious problem in the establishment and long-term viability of primary sponge cell cultures (Rinke­ vich, 1999). Although precautions taken against microbial contaminants by the addition of antibiotics and antifungal agents is generally successful, resistance to these antimicrobials by some micro-organisms remains a possibility. A further, equally serious problem is the difficulty of differentiating sponge cells from some unicellular prokaryotic and eukaryotic contaminants in culture. Sponge cells are extremely variable morphologically, and a majority of the marine micro­ organisms encountered in cultures are unknown. It is fundamentally important to develop consistent tools - molecular or otherwise - for the specific identification of sponge cells in culture. Muller et al. (1999) reported the identification and isolation of a specific gene from Suberites domuncula which they have used to probe and compare RNA extracted from primmorphs with that of the source sponge. Of course, verifying the identity of cell cultures (versus primmorphs) to rule out the presence of contaminants-is'tnoreproblematic. It is not sufficient to verify that cells of-the source sponge are in culture. One must also verify that cells of potential contaminants are not present. This involves development not only of a specific sponge probe, but also probes for potential eukaryotic contaminants, such as yeasts and thraustochytrids. An alternative approach one may use to verify identity of cultured sponge cells is expression of production of sponge metabolites. Although this may be considered an indirect verification of identity, it is nonetheless valid, particularly if the metabolite produced in culture is a major metabolite of the source sponge. One may also reliably assess metabolic activity (e.g. DNA synthesis, protein synthesis and enzyme activity) of sponge cells in 'high-throughput mode' (Willoughby and Pomponi, 2000) - particularly when evaluating the effects of matrices of variables on cell culture - in much the same way variables are tested in mammalian cell Sec. 14.7] Current status and Future directions 333 cultures (i.e. using microwell assays). If contamination is suspected, molecular probes for prokaryotic and eukaryotic contaminants, as well as sponge-specific probes as described by Muller et a/. (1999), may be applied for confirmation of identity.

14.6.1 Chemical analyses of cultured cells The ability to monitor presence and production of bioactive metabolites in sponge cell cultures is essential for optimizing culture conditions. Stevensine is a major / metabolite of Teichaxinella morchella (and related sponge taxa) (Albitzi and Faul- kner, 1985) and is not likely to be produced by associated micro-organisms (see discussion above). We have demonstrated that cell cultures which underwent cell division in response to PHA continued to produce stevensine in culture (Pomponi et a/., 1997b, 1998). These results indicate not only that the cells retain their ability to synthesize stevensine after doubling, but they also demonstrate that the methods we have developed for dissociation, culture and mitogenic stimulation do not inhibit cell division, or secondary metabolite-biosynthetic pathways.

14.6.2 Biosynthesis of radiolabelled precursors Biosynthesis of stevensine in primary cultures of Teichaxinella morchella was further verified by incubation of enriched archaeocytes with radiolabelled amino-acid pre­ cursors and subsequent detection of incorporation of the precursors into stevensine (Andrade et a/., 1999). J4C-histidine, 14C-ornithine and 14C-proline were used in the biosynthesis of radioactive stevensine. This provides further evidence to support both the identity of these primary cultures as well as their ability to continue to produce characteristic second ary metabolites after dissociation, cryopreservation, thawing and primary culture.

14.7 CURRENT STATUS AND FUTURE DIRECTIONS

We have developed a basal nutrient medium for primary eulrure'of dissociated cells. The supplementation of this medium with exogenous growth factors and lectins resulted in increased DNA content and cell division in primary cultures. We have demonstrated that primary cultures of sponge cells dissociated using our techniques will continue to synthesize DNA, divide and biosynthesize secondary metabolites found in the source sponge. The results of preliminary scale-up experiments indicate that sponge cells can be cultured in suspension. All of these results support the potential for development of an in vitro production system for sponge-derived bioactive compounds, as well as a model system to study basic metabolic processes involved in cell differentiation. Research continues in our laboratory on the identi­ fication and characterization of cell-cycle-regulating genes, growth-factor genes and their genetic control mechani sms and promoters; the development of a normal or transformed cell line, and the identification ofgenes that are involved in biosynthesis 334 Development of sponge cell cultures for biomedical applications [eh.14 of bioactive metabolites. This research may ultimately facilitate our ability to manip­ ulate or overexpress cell proliferation and bioactive metabolite production in normal or transformed sponge cell lines.

14.8 ACKNOWLEDGEMENTS

This research was funded in part by grants from the National Institutes of Health, the National Sea Grant Marine Biotechnology Program and the Florida Sea Grant College Program. We acknowledge the contributions of our colleagues at Harbor Branch in development of sponge cell-culture techniques: Drs Amy E. Wright, Susan H. Sennett and Jose V. Lopez. This is Harbor Branch Oceanographic Institution Contribution Number 1343.

14.9 REFERENCES

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